We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. ...The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.
We developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. As an example, we compared Iba-1 ...(microglial marker) positive cell densities in rats with (
n
=
6) and without (
n
=
6) unilateral injection of 1-methyl-4-phenylpyridinium salt (MPP
+). Immunopositive cells were automatically counted in each animal over a coronal section in the midbrain (bregma −5.9
mm) and a positive cell density map was created for each animal. After the positive cell density map was normalized to a template section from an atlas, positive cell densities of the two groups were compared in each pixel over the section and a statistical parameter (
p-value from
t-test) was mapped on each pixel. We were able to detect significant increases of microglias in the side of MPP
+ injection not only in the substantia nigra pars compacta but also in adjacent white matter. We also applied the same analysis to tyrosine hydroxylase stained sections and detected significant decreases of dopamine neurons in the side of MPP
+ injection. The new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Aim
Although it is accepted that progesterone (P) induces acrosome reaction through non-genomic regulation, it is not well known if P also affects hyperactivation of sperm.
Methods
Hamster ...spermatozoa were hyperactivated by incubation for 4 h on modified Tyrode’s albumin lactate pyruvate medium and recorded on a DVD via a charge-coupled device camera attached to a microscope with phase-contrast illumination and a small CO
2
incubator. Phosphorylation of proteins was detected by western blotting using antiphosphotyrosine antibodies.
Results
Sperm hyperactivation was significantly increased and accelerated by a non-genomic signal of P. Although acceleration of motility of hyperactivated sperm occurred with 10, 20 and 40 ng/mL P, the most effective concentration was 20 ng/mL. Progesterone also significantly increased 80-kDa tyrosine phosphorylation of sperm proteins. Both extracellular Ca
2+
and albumin were essential for sperm hyperactivation, and the former was also essential for maintaining sperm flagellar movement. Moreover, phospholipase C (PLC) was associated with the regulation of hyperactivation by P.
Conclusion
It is likely that P regulates sperm hyperactivation by a non-genomic signal in relation to tyrosine phosphorylation and PLC.
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DOBA, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Peptidoglycan recognition proteins (PGLYRPs) are a family of pattern recognition receptors (PRRs) that are able to induce innate immune responses through their binding to peptidoglycan (PGN), ...lipopolysaccharide, or lipoteichoic acid, or by interacting with other PRR-ligands. Recently, progress has been made in understanding the immunobiology of PGLYRPs in human and mice, however, their functions in livestock animals have been less explored. In this study, we characterized the expression patterns of PGLYRPs in porcine intestinal epithelial (PIE) cells and antigen-presenting cells (APCs) and their modulation by the interactions of host cells with PRR-ligands and non-viable immunomodulatory probiotics referred to as paraimmunobiotics. We demonstrated that PGLYRP-1, -2, -3, and -4 are expressed in PIE cells and APCs from Peyer's patches, being PGLYPR-3 and -4 levels higher than PGLYRP-1 and -2. We also showed that PGLYRPs expression in APCs and PIE cells can be modulated by different PRR agonists. By using knockdown PIE cells for TLR2, TLR4, NOD1, and NOD2, or the four PGLYRPs, we demonstrated that PGLYRPs expressions would be required for activation and functioning of TLR2, TLR4, NOD1, and NOD2 in porcine epitheliocytes, but PGLYRPs activation would be independent of those PRR expressions. Importantly, we reported for the first time that PGLYRPs expression can be differentially modulated by paraimmunobiotic bifidobacteria in a strain-dependent manner. These results provide evidence for the use of paraimmunobiotic bifidobacteria as an alternative for the improvement of resistance to intestinal infections or as therapeutic tools for the reduction of the severity of inflammatory damage in diseases in which a role of PGLYRPs-microbe interaction has been demonstrated.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ...ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
A novel magnetic separation method, which utilizes the magneto-Archimedes levitation, has been introduced and applied to separation of biological materials. By using the feature that the stable ...levitation position under a magnetic field depends on the density and magnetic susceptibility of materials, we have successfully separated biological materials such as hemoglobin, fibrinogen, cholesterol, and so on. So far, the difference of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation seems to be a potential way in biological materials separation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
Background: Despite the approval of immune checkpoint inhibitors (ICIs), prognosis of small cell lung cancer (SCLC) remains poor. DLL3 is upregulated in SCLC whereas expression in normal ...tissues is minimal, representing the favorable profile as a therapeutic target. T cell engager (TCE) is a potent immunotherapy that redirects T cells to tumors expressing a specific antigen. Unlike ICIs, TCEs do not require the recognition of tumor antigens by T cells and thus could be an alternative approach to target tumors where the benefit of ICIs is limited such as SCLC. CD137 (4-1BB) is a costimulatory molecule that promotes T cell activation, proliferation, and survival. Since CD137 agonists synergize with CD3-mediated T cell activation, inducing concurrent CD137 costimulation is a promising strategy to unleash the potential of TCEs. We therefore developed a DLL3/CD3/CD137 trispecific T cell engager composed of two CD3/CD137 dual specific Fabs and one extra DLL3 Fab (DLL3 trispecific, RG6524).
Results: We initially investigated the CD3 and CD137 signal transduction in Jurkat cells harboring NFAT or NF-κB reporter cocultured with DLL3 positive cells. DLL3 trispecific showed dose-dependent increase in NFAT and NF-κB activity, indicating that target engagement successfully activated CD3 and CD137 signaling. In the in vitro assay using human PBMC, DLL3 trispecific induced cytotoxicity against SCLC cell lines with EC50s in the two-digit pM range.
In vivo efficacy was evaluated in SCLC xenograft models established by engrafting SCLC cell lines into immune humanized NOG mice. DLL3 trispecific showed greater tumor growth inhibition compared to a traditional bispecific T cell engager. Furthermore, flow cytometry-based immunophenotyping revealed that DLL3 trispecific increased T cell number in tumors and improved IFN-γ production from CD8 T cells. We also explored the combination with platinum-based drugs, which are widely used for SCLC, and showed that DLL3 trispecific enhanced the efficacy of platinum-based drugs. These data demonstrated that DLL3 trispecific has the potent in vivo efficacy and the potential for clinical use.
We next evaluated whether cytokine release syndrome (CRS) mitigating approaches affect antitumor efficacy. Although prophylactic use of a steroid significantly reduced cytokine production, tumor growth inhibition by DLL3 trispecific remained unchanged. Likewise, tocilizumab treatment did not reduce the efficacy, suggesting that CRS mitigation did not abrogate the therapeutic benefit.
We finally assessed the tolerability in non-human primates. DLL3 trispecific did not reach the maximum tolerated dose and was well tolerated up to 16 mg/kg Q2D, the highest dose tested.
Conclusion: Our data showed that DLL3 trispecific has potent activity and is well suited for clinical application in SCLC. These findings provide a rationale for the clinical testing of DLL3 trispecific.
Citation Format: Hirofumi Mikami, Shu Feng, Sotaro Naoi, Yumiko Azuma, Yoko Kayukawa, Toshiaki Tsunenari, Kentaro Asanuma, Ryutaro Iwabuchi, Hiroaki Nagano, Junko Shinozuka, Masaki Yamazaki, Haruka Kuroi, Siok Wan Gan, Priyanka Chichili, Shun Shimizu, Yutaka Matsuda, Shinya Ishii, Shogo Kamikawaji, Yasuko Kinoshita, Yuichiro Shimizu, Akihisa Sakamoto, Masaru Muraoka, Noriyuki Takahashi, Tatsuya Kawa, Hirotake Shiraiwa, Kenji Kashima, Futa Mimoto, Mika Kamata-Sakurai, Takehisa Kitazawa, Tomoyuki Igawa. A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1872.
Expression of immediate early genes, such as c-fos, has been extensively used as a marker of neural activity. However, their expression in the brain has so far been examined by using invasive ...procedures. In this study, we tried to image c-fos expression in the mouse barrel cortex noninvasively by detecting bioluminescence produced by the reporter luciferase. To detect asymmetry in c-fos expression in the bilateral barrel cortices, we used ten Fos-Luc mice and removed long whiskers on one side. After 1h of exploration in a novel cage, luciferin was intraperitoneally administrated under gas anesthesia and bioluminescence was measured with a cooled CCD camera. We observed moderate but clear emission over the head that was significantly stronger on the side of removal. After regrowth of the whiskers, the same mice had the vibrissae clipped on the other side. Bioluminescence was again dominant on the side of removal. In three of the mice, c-fos expression was examined immunohistochemically. The distribution of bioluminescence generally agreed with that of the c-fos positive cells though the bioluminescence tended to distribute wider, by around 0.5mm, probably due to scattering of light through the tissues. The results show that expression of c-fos in the mouse barrel cortex can be imaged repeatedly and noninvasively in the living animal.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Magnetic fields up to 10
T have been applied on various substances composed of non-magnetic liquids, solids and/or gases. It has turned out that the magnetic fields of this range do produce various ...visible effects on the equilibrium shape, relative distribution of the substances or kinetic processes of the systems. The phenomena observed are due to the magnetization force that becomes non-significant in determining the mechanical balance of the system. The effects manifest themselves through the deformation of the equilibrium shape of the liquid interfaces, through the change in the effective weight which determines the relative positions occupied in the space by the substances involved and through the creation of convection in a non-uniform gas or liquid phase in terms of the magnetic susceptibility. Some of the processes seem to be utilized for practical purposes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter ...assay using a toll-like receptor 2-expressing transfectant (HEKpTLR2 system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEKpTLR2 and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEKpTLR2 system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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